A common technique for cloning receptors is to use nucleic acid hybridization technology to identify receptors which are homologous to other, known receptors. For instance, originally the cloning of seven transmembrane domain G protein-coupled receptors (GPCR) depended on the isolation and sequencing of the corresponding protein or the use of expression cloning techniques. However, when sequences for these receptors became available, it was apparent that there were significant sequence homologies between these receptors. Because this technology does not require that the ligand of the receptor be identified, the cloning of a large number of “orphan receptors”, which have no known ligand and the biological function of which is often obscure, has resulted. Receptors of all types comprise this large family.
Known orphan receptors include the nuclear receptors COUP-TF1/EAR3, COUP-TF2/ARP1, EAR-1, EAR-2, TR-2, PPAR1, HNF-4, ERR-1, ERR-2, NGFIB/Nur77, ELP/SF-1 and MPL (Parker et al. supra, and Power et al. (1992) TIBS 13:318-323). A large number of orphan receptors has been identified in the EPH family (Hirai et al. (1987) Science 238:1717-1720). HER3 and HER4 are orphan receptors in the epidermal growth factor receptor family (Plowman et al. (1993) Proc. Natl. Acad. Sci. USA 90:1746-1750). ILA is a newly identified member of the human nerve growth factor/tumor necrosis factor receptor family (Schwarz et al. (1993) Gene 134:295-298). IRRR is an orphan insulin receptor-related receptor which is a transmembrane tyrosine kinase (Shier et al. (1989) J. Biol Chem 264:14606-14608). Several orphan tyrosine kinase receptors have been found in Drosophila (Perrimon (1994) Curr. Opin. Cell Biol. 6:260-266). The identification of ligands for orphan receptors is important to drug discovery.
One large subgroup of orphan receptors, as alluded to above, is found in the G protein coupled receptor (GPCR) family. Approximately 100 such receptors have been identified as mediators of transmembrane signaling from external stimuli (vision, taste and smell), endocrine function (pituitary and adrenal), exocrine function (pancreas), heart rate, lipolysis, and carbohydrate metabolism. Structural similarities suggest that the G protein-coupled receptors of animals can be subclassified into three distinct groups: (i) the largest class including monoamine, cytokine, lipid, neuropeptide etc. receptors; (ii) the class represented by calcitonin, secretin and VIP receptors but also containing orphan receptors like emr-1 (Baud V. et al., 1995 Genomics 26: 334-44) and methuselah (Lin Y. J. et al., 1998 Science 282: 943-6); and (iii) the metabotropic glutamate and calcium-sensing receptors.
Formyl peptide receptor like-1 receptor (FPRL-1) was identified as an orphan GPCR through low stringency hybridization of a human formyl peptide receptor (FPR1)-specific cDNA probe to a cDHA library derived from HL-60 cells (Murphy, et al. (1992) J. Biol. Chem. 267:7637-7643; Ye, R. D., et al. (1992) Biochem. Biophys. Res. Comm. 184:582-589). FPRL-1-specific RNA is expressed in neutrophils and monocytes (Durstin, et al. (1994) Biochem. Biophys. Res. Comm. 201:174-179). The receptor exhibits 69% amino acid identity to FPR1 and maps to the locus on human chromosome 19 that contains the genes for the C5a receptor, FPR1 and for a second FPR1-related orphan, FPRL-2 (Bao, et al. (1992) Genomics 13:437-440). FPR1 is also expressed in neutrophils and monocytes and is stimulated by N-formylated peptides of bacterial origin. Specific binding of the ligand fMLP to FPR1 on neutrophils stimulates calcium mobilization and results in a variety of cellular changes including chemotaxis, degranulation and the respiratory burst. FPRL-1 has been characterized as a low affinity receptor for fMLP (Ye, et al. (1992) Biochem. Biophys. Res. Comm. 184:582-589) and a high affinity receptor for lipoxin A4 (Fiore, et al. (1994) J. Exp. Med. 180:253-260). However, treatment of cells expressing FPRL-1 with lipoxin A4 results in a limited biological response (Fiore, et al. (1994) J. Exp. Med. 180:253-260), so the role of this receptor in the normal functioning of neutrophils and monocytes remains unresolved.
Previous work describes the expression of recombinant mammalian G protein-coupled receptors as a means of studying receptor function in order to identify agonists and antagonists of those receptors. For example, the human muscarinic receptor (HM1) has been functionally expressed in mouse cells (Harpold et al. U.S. Pat. No. 5,401,629). The rat V1b vasopressin receptor has been found to stimulate phosphotidyinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary cells upon agonist stimulation (Lolait et al. (1995) Proc Natl. Acad Sci. USA 92:6783-6787). These types of ectopic expression studies have enabled researchers to study receptor signaling mechanisms and to perform mutagenesis studies which have been useful in identifying portions of receptors that are critical for ligand binding or signal transduction.
Experiments have also been undertaken to express functional G protein coupled receptors in yeast cells. For example, U.S. Pat. No. 5,482,835 to King et al. describes a transformed yeast cell which is incapable of producing a yeast G protein α subunit, but which has been engineered to produce both a mammalian G protein α-subunit and a mammalian receptor which is “coupled to” (i.e., interacts with) the aforementioned mammalian G protein α-subunit. Specifically, U.S. Pat. No. 5,482,835 discloses expression of the human beta-2 adrenergic receptor (β2AR), a seven transmembrane receptor (STR), in yeast, under control of the GAL1 promoter, with the β2AR gene modified by replacing the first 63 base pairs of coding sequence with 11 base pairs of noncoding and 42 base pairs of coding sequence from the STE2 gene. (STE2 encodes the yeast α-factor receptor). King et al. found that the modified β2AR was functionally integrated into the membrane, as shown by studies of the ability of isolated membranes to interact properly with various known agonists and antagonists of β2AR. The ligand binding affinity for yeast-expressed β2AR was said to be nearly identical to that observed for naturally produced β2AR.
U.S. Pat. No. 5,482,835 also describes co-expression of a rat G protein α-subunit in the same cells, yeast strain 8C, which lacks the cognate yeast protein. Ligand binding resulted in G protein-mediated signal transduction. U.S. Pat. No. 5,482,835 further teaches that these cells may be used in screening compounds for the ability to affect the rate of dissociation of Gα from Gβγ in a cell. For this purpose, the cell further contains a pheromone-responsive promoter (e.g., BAR1 or FUS1), linked to an indicator gene (e.g. HIS3 or LacZ). The cells are placed in multi-titer plates, and different compounds are placed in each well. The colonies are then scored for expression of the indicator gene.
Genome sequencing efforts and homology-based cloning have revealed a large number of human genes encoding G protein-coupled receptors (GPCRs) of unknown function. Elucidation of the function of these orphan receptors has been difficult, relying primarily on homology to known receptors, circumstantial inference from expression patterns or identification of the natural ligand for the receptor. This latter process, although successful in identifying anadamide as a potential endogenous ligand of the cannabinoid receptor (Devane, et al. (1992) Science 258:1946-1949) and the pituitary neuropeptide nociceptin as an agonist of the opioid-like GPCR, ORL 1 (Meunier, et al. (1995) Nature 377:532-535), is inherently inefficient, involving methodical searches through extracts of likely tissue sources.